Crystal Surface Functionalisation

QCM measurements used a quartz crystal with a top and bottom gold electrode. To enable detection

the gold surface needed to be functionalised with a capture element. This element would selectively

bind the analyte of choice to the electrode surface. A complementary nucleic acid sequence was used

as the capture element, this means all nucleic acids between the capture element and analyte base

pairs matched (see figure 42). The use of a complementary sequence as a capture element has been

shown in literature [397-399]. The element was attached to the gold surface using thiols, as they form

a self-assembled monolayer on gold [565, 566]. Two functionalisation strategies were used: thiolated

109 The thiolated DNA surface consisted of the complementary miRNA sequence with thymine bases

added between the thiol and the complementary sequence. The addition of the thymine bases adds

a spacer and hydrophilic section which has been shown to prevent non-specific binding [567]. The

thiolated DNA was then functionalised with a second thiol mercaptohexanol (MCH). The Figure 42: Surface functionalisation of QCM crystals both thiolated DNA and biotinylated thiol

functionalisation. a) is a blank crystal, b) addition of thiolated DNA, c) back filling crystals with MCH, d) Detection with ssDNA analyte. Biotin thiol functionalisation corresponds to, f) after incubation with biotin thiol

and methoxy thiols, g) addition of streptavidin, h) biotinylated DNA functionalisation and i) detection with ssDNA analyte. e) depicts the sequences used in the thiolated DNA functionalisation assay, the black sequence

in the thiolated DNA attached to the crystal surface and the red sequence is the analyte ssDNA. J) depicts the sequences involved in the biotin DNA assay, the black sequence is the biotinylated DNA and the red sequence

110 functionalisation of the second thiol had two advantages. One is that nitrogen containing bases in the

DNA can form self-assembled monolayers with gold, however the affinity is not as high as sulphur,

therefore the DNA sequences may lie parallel to the surface, the functionalisation with the second

thiol will prevent this [568]. Secondly, the thiol is a way to control the density of thiolated DNA

sequences on the surface. The literature suggests that a lower density of the complementary

sequence leads to an increase in hybridisation efficiency as it lowers the steric hinderance [569]. The

thiolated DNA capture element is 9 nm long, therefore is well within the roughly 480 nm penetration

depth in water (calculated from the equathion located in [570]).

An alternative functionalisation strategy uses biotinylated thiols, the full functionalisation procedure

is outlined in Figure 42. Previous studies have been conducted comparing thiolated DNA and

biotinylated thiol functionalisation techniques and found biotin to be more efficient in detecting DNA

due to higher hybridisation efficiencies [571, 572]. However, these papers utilised carboxylic acid

modified thiols and EDC/NHS chemistry to attach streptavidin rather than biotinylated thiols. When

comparing the use of carboxylic thiols with EDC/NHS and biotinylated thiols, biotinylated thiols were

found to give a more ordered streptavidin surface and therefore a more ordered attachment of the

complementary sequence leading to higher hybridisation efficiencies [573]. The biotin thiol surface

was co-functionalised with methoxy thiols, this lowers the density of biotinylated thiols and therefore

lowers the steric hindrance. Both methoxy and biotinylated thiols contained ethylene glycol units,

which has shown to lower non-specific binding of proteins [574]. The length of the biotinylated thiol

(roughly 5 nm calculated from bond lengths), streptavidin (roughly 6 nm, obtained from [575]) and

biotinylated DNA (9 nm) was roughly 20 nm, this is well within the penetration depth of the 14.3 MHz

crystal.

5.2.3.1 Biotin thiol assay

The crystal was cleaned as outlined in section 5.2.2 then incubated in an absolute ethanol solution

111 chosen through evidence from literature [410]). The crystal was washed then assembled into a flow

cell. After assembly the crystal was further functionalised with 10 µg/mL of streptavidin for 30 min,

then 1 µM biotinylated DNA for 30 min all with a flow rate of 10 µL/min. Detection was conducted

using the complementary DNA sequence for 30 min. The assay was conducted in 10 mM PBS. (used a

syringe pump set to withdraw from an Eppendorf tube, through the crystal set-up then into a syringe.

Pump obtained from Harvard Apparatus).

5.2.3.2 Thiolated DNA assay

Before functionalisation crystals were cleaned as outlined in section 5.2.2. Two buffer solutions were

used for functionalisation as during experiments using the two buffers was found to give a better

result. The hybridisation buffer (for binding of analyte ssDNA to the capture element) and

immobilisation buffer (for functionalisation of thiolated DNA to the gold surface of the crystal).

Immobilisation buffer contained 1x TE buffer, 1 M NaCl, 50 mM MgCl2 and 10 mM EDTA and the

hybridisation buffer was 1x TE, 1 M NaCl and 0.1 M MgCl2.

Thiolated DNA was reduced with 0.5 M TCEP in a 1:1 (v/v) ratio for 1-2 hours at room temperature.

After reduction the DNA was diluted to 1 µM in immobilisation buffer. The crystal was cleaned and

assembled into the flow cell, the reduced DNA was flowed over the crystal for 30 min, then 1 mM

MCH diluted in immobilisation buffer for 30 min. For detection hybridisation buffer was flowed until

baseline stabilisation, the complementary sequence was then diluted in hybridisation buffer and

flowed over the crystal for 30 mins, the assay was conducted at flow rate of 10 µL/min (used a syringe

pump set to withdraw from an Eppendorf tube, through the crystal set-up then into a syringe. Pump

obtained from Harvard Apparatus).

Thiolated DNA was also functionalised to the surface while a potential was applied to the gold

electrode on the crystal. The functionalisation under potential involved incubating the crystal in the

reduced thiolated DNA solution while assembled in the electrochemical flow cell under a voltage of

112 aqueous MCH for 30 min and washed again. For detection the crystal was washed in hybridisation

buffer three times and scans taken before hybridisation, then the complementary sequence was

spiked in hybridisation buffer and left for 30 min. This assay was not conducted under flow using a

syringe pump as the assay used the electrochemical set-up, therefore baseline stabilisation was not

conducted, multiple scans were taken and then averaged.